The present invention relates to polypeptide agonists of the human cytokine interleukin 8 to other human alpha chemokines and to the method of using these agonists.
Interleukin 8 (IL-8) is a human cytokine that promotes the recruitment and activation of neutrophil leukocytes and represents one of several endogenous mediators of the acute inflammatory response. In the past it was variously termed neutrophil-activating factor, monocyte-derived neutrophil chemotactic factor, interleukin-8 (IL-8), and neutrophil-activating peptide-1. IL-8 has gained the widest acceptance and will be used herein.
The most abundant naturally occurring form of the IL-8 monomer is a 72-residue protein apparently derived by processing of a 99-residue precursor. Other proteins with related sequences, including neutrophil-activating peptide-2 and GRO.alpha. (with melanoma growth stimulatory activity) are IL-8 homologues which have neutrophil-activating properties.
IL-8 is a member of the chemokine superfamily that is divided into two distinct function classes: alpha (.alpha.) and beta (.beta.). The members of each class share an organizing primary sequence motif. The .alpha. members are distinguished by a C-X-C motif with the first two cysteines in the motif separated by an intervening residue. C-X-C chemokines are potent chemoattractants and activators for neutrophils, and are represented by IL-8. The .beta. family chemokines have a C--C motif and are equally potent chemoattractants and activators of monocytes. It appears that the two sides of the chemokine family have clearly defined functions: the C-X-C subfamilies cannot activate monocytes while the C--C subfamily has no effect on neutrophils. Nonetheless these two families of chemokines have similar structures although fairly low sequence homology (30 to 35%). Proteins within the same family such as platelet factor four (PF-4) are structurally related to IL-8 (35% sequence identity) but lack the N terminal ELR sequence (Glu-Leu-Arg) which has been shown by site directed mutagenesis to be critical for IL-8 activity and thus, PF-4 has an entirely different profile of activity. Indeed, when the ELR sequence is added to the N-terminus of PF-4 it has been found that the modified protein has potent neutrophil activation and chemoattractant properties (Clark-Lewis, I; Dewald, B.; Geiser, T.; Moser, B.; Baggiolini, M.: Platelet factor 4 binds to interleukin 8 receptors and activates neutrophils when its N terminus is modified with Glu-Leu-Arg. Biochemistry 90:3574-3577, 1993). However this is not true for all of the chemokines since two of the proteins related to IL-8, .gamma. interferon inducible protein (IP-10) and monocyte chemoattractant protein 1 (MCP-1) do not acquire neutrophil activating properties when the ELR structural determinants are added.
In studies by Clark-Lewis (Clark-Lewis, I., et al.: Structural requirements for interleukin-8 function identified by design of analogs and CXC chemokine hybrids. J Biol Chem 269:16075-16081, 1994) it was shown that conservative substitutions are accepted into the 10-22 region of IL-8 in contrast with the ELR motif (residues 4-6). They concluded that the disulfide bridges and the 30-35 turn provide a structural scaffold for the NH-2 terminal region which includes a primary receptor binding site (ELR motif) and secondary binding and conformational determinants similar to those seen in residues 10 through 22. Other studies using mutants of IL-8 and melanoma growth stimulating activity (MGSA) and recombinant IL-8 .alpha./.beta. receptors stably expressed in human cells demonstrated that there was a second site on the molecule responsible for binding. It appears that the carboxy terminus distal to amino acid 50 is not important in high affinity binding to the .alpha. receptor although both the amino and carboxy termini appear to be important for binding to the .beta. receptor (Schraufstatter, I.S., et al., Multiple sites on IL-8 responsible for binding to .alpha. and .beta. IL-8 receptors. J Immunol 151:6418-6428, 1993). In summary, it appears that there are at least two and maybe three regions responsible for binding on IL-8. Further, the specific contact pharmacophore may vary depending upon whether or not the .alpha. or the .beta. receptor is being examined.
The in vitro effects of IL-8 on neutrophils are similar to those of other chemotactic agonists such as C5a and fMet-Leu-Phe and include induction of a transient rise in cytosolic free calcium, the release of granules containing degradative enzymes such as elastase, the respiratory H.sub.2 O.sub.2 burst, neutrophil shape change, and chemotaxis. IL-8 appears to bind to at least one class of receptor sites on neutrophils with a frequency of approximately 64,000/cell and a K.sub.d of 0.2 nM.
The three-dimensional structure of IL-8 is known by two-dimensional NMR and x-ray diffraction techniques. The IL-8 monomer has antiparallel .beta. strands followed by a single overlying COOH-terminal .alpha. helix. Two disulfide bridges, between cysteines 7 and 34, and between cysteines 9 and 50 seem to stabilize the tertiary structure. Residues 1-6 and the loop residues 7-18 seem to have little defined secondary structure. In solution, IL-8 is a noncovalent homodimer which is stabilized primarily by interactions between the .beta. strands of the two monomers.
Examination of the three-dimensional structure indicates that following the cysteine at position 50, the residues form a type 1 .beta. turn (at residues 51 to 55) followed by an amphipathic a helix (at residues 55 to 72) that transverses the .beta. sheet. The hydrophobic face of the .alpha. helix interacts with and stabilizes the hydrophobic face of the .beta. sheet. Some of the interactions are between the two subunits of the dimeric molecule.
Interleukin-8 has shown both anti-tumor and anti-infective therapeutic activity. IL-8 has been shown to induce the regression of macroscopic tumors in a model of peritoneal carcinomatosis in the rat. In this model IL-8 was shown to recruit PMN to the challenge site but did not enhance PMN infiltration of the tumor or the cytotoxic activity of PMN. Regardless, it did have significant therapeutic activity which may be secondary to PMN cytotoxicity and associated with other intermediate cells. It is suggested that lymphocytes could be involved since IL-8 has also demonstrated the ability to stimulate T-cell chemotaxisis. (Lejeune, P., et al.: Interleukin-8 has antitumor effects in the rat which are not associated with polymorphonuclear leukocyte cytotoxicity. Cancer Immunol Immunotherapy 38:167-170, 1994). Similarly, Interleukin-8 has shown therapeutic activity in nonneutropenic mice who received IL-8 shortly before challenge and at the site of infectious challenge with either P. aeruginosa, Klebsiella-phenumoniae, or Plasmodiumberghei. (Vogels, M. T., et al., Effects of Interleukin-8 on nonspecific resistance to infection in neutropenic and normal mice. Antimicrob-Agents-Chemother 37:276-280, 1993).
In other antitumor studies, IP-10, an alpha chemokine whose secretion is induced by IFN-.gamma. and LPS, was genetically engineered into tumor cells. The expression of IP-10 by several tumor cell lines had no effect on the growth of these tumor cells in vitro but elicited a powerful host mediated anti-tumor effect in vivo. Indeed, tumors genetically engineered to secrete IP-10 elicited a T-lymphocyte dependent anti-tumor response resulting in the rejection of tumors in vivo. Animals injected with these tumor cells do not develop tumors or develop tumors which spontaneously regress. Further, tumors induced with the parent tumor cells admixed with IP-10 secreting tumor cells protect the animals against subsequent growth . This suggests that in addition to being chemotactic for T-cells (Clark-Lewis, I., et al., Structural requirements for Interleukin-8 function identified by design of analogs and CXC chemokine hybrids. J Biol Chem 269:16075-16081, 1994) alpha chemokines may also act as T-cell adjuvants and therapeutics via T-cell chemotaxis and/or augmentation (Luster, A. D., a CXC chemokine, elicits a potent thymus-dependent antitumor response in vivo. J Exp Med 178:1057-1064, 1993).
IL-8 has been previously produced through chemical synthesis (for example see: Clark-Lewis, et al., "Chemical Synthesis, Purification, and Characterization of Two Inflammatory Proteins; Neutrophil-Activating Peptide-1 (Interleukin-8) and Neutrophil-Activating Peptide-2" (1991) Biochemistry 30: 3128-3135) and by recombinant DNA methods (for example see: Herbert, et al., "Scanning Mutagenesis of Interleukin-8 Identifies A Cluster of Residues Required for Receptor Binding" (1991) J. Biol. Chem. 286: 18989-18994). In addition, it is known that IL-8 exists in several forms that vary at the NH.sub.2 -terminus, which have been detected in preparations purified from natural sources. These variations correspond to the predominant 72-residue form (which is generally considered to be the prototype IL-8 molecule); a 77-residue form having 5 additional NH2-terminus amino acids on each monomer; and, two shortened forms having residues 3-72 and 4-72 of the 72 amino acid form, respectively.